The long-term objective of our laboratory is to understand the regulatory roles of TFPI-2 in the invasive and malignant behavior of glioblastomas. TFPI-2 inhibits six different serine proteases, including plasmin, an enzyme that appears to be directly involved in the invasive behavior of primary tumors, particularly glioblastomas. In preliminary studies, TFPI-2 was undetectable in both glioblastomas and an established glioblastoma cell line that is highly invasive in vitro and in vivo. In contrast, the TFPI-2 protein was detected in normal human brain and, to a lesser extent, in low-grade gliomas and anaplastic astrocytomas as well as in cell lines derived from these tumors. These findings show an inverse correlation between the TFPI-2 levels and the progression of gliomas. We postulate that modulation of TFPI-2 expression will alter the invasive behavior of gliomas in vitro and in vivo. The central hypothesis is that TFPI-2 is a key negative regulator of proteases that promote glioma invasion and angiogenesis. To test this hypothesis, first we examine Specific Aim 1: Construct an expression vector cassette containing a 0.7-kb fragment of human TFPI-2 in the sense orientation in an E1-deleted region driven by an independent cytomegalovirus (CMV) promoter and the polyadenylation signal bovine growth hormone (BGHpA) and study its (Ad-TFPI-2) effect on the levels of TFPI-2, other proteases in glioma cells and their invasive behavior both in vitro and in vivo models and its effect on glioma growth in vivo. 1a) Construct and determine the effect of the Ad-TFPI-2 construct on the levels of TFPI-2 and proteases, 1b) Determine the effect of the Ad-TFPI-2 construct on glioma cell growth, adhesion and migration with that of mock and Ad-CMV construct, 1c) Investigate the effect of the TFPI-2 construct on the invasive behavior of human glioma cells in vitro models, 1d) Determine the effect of Ad-TFPI-2 construct to inhibit the invasion and growth of human glioma cell lines injected subcutaneously and intracerebrally in nude mice. 1e) Evaluate the toxicity of Ad-TFPI-2 construct with that of Ad-CMV construct given as intracerebral injections.
Specific Aim 2 : Identify the molecular mechanisms that regulate cerebral angiogenesis in relation to the production of TFPI-2 in co-cultures of endothelial and glioma cells both in vitro and in vivo. 2a) Determine that levels of TFPI-2, VEGF, TF and other proteases and the length of capillary-like structures formed during cocultures of endothelial cells with sense/antisense TFPi-2 stable clones or glioblastoma cell lines and primary glioblastoma cells infected with Ad-CMV and Ad-TFPI-2 sense constructs or in the presence of an inhibitor and 2b) Determine the effect of sense and antisense stable transfectants or infection of Ad-TFPI-2 sense construct or in the presence of an inhibitor specified in specific aim 2a on tumor angiogenesis. We believe that by determining the role of TFPI-2 in the invasiveness of glioblastomas and its role in angiogenesis, it will be possible to design further studies that will evaluate the therapeutic role of this novel protease inhibitor in brain tumors.
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